(1) Field of the Invention
The present invention relates to a 5xe2x80x2 upstream region DNA containing a promoter of a human bone morphogenetic protein (hereinafter called BMP-2). Further, the present invention provides a method for exploring a low molecular weight compound positively or negatively which regulates the expression of human BMP-2 by using a mass of animal cells that are introduced with a 5xe2x80x2 upstream region DNA containing the human BMP-2 promoter and a structure connected to a suitable reporter gene, and by using a reporter activity as an indicator.
(2) Description of the Related Art
Many substances have been known as usable for healing of bone deficit, fracture, etc. by inducing bone regeneration. Among them, BMP is a protein belonging to TGF (transforming growth factor) -xcex2 superfamily and has been formerly found as a substance inducing ectopic ossification and existing in a decalcified product of a bone (Trends in Biotechnol. 11: 379-383, 1993). BMP members known so far range from BMP-1 to BMP-14. Among them, the members from BMP-2 to BMP-14 have been known as showing the bone morphogenetic activity. BMP-2 has been known as having the strongest activity of bone morphogenesis (Science 242: 1528-1534). It has been known that recombinant BMP-2 induces ectopic ossification (Proc. Natnl Acad. Sci. USA 87: 2220-2224, 1990), and is effective to bone deficit in animal model (J. Bone and Joint Surg. 74A, 659-670, 1992). The BMP proteins, particularly ranging from BMP-2 to BMP-14, are effective to therapeutic and preventive treatments of various bone disturbances and bone diseases. However, the BMP proteins exist in very small quantity in nature, and for an available large quantity for therapeutic use, production of a recombinant protein is necessary. The production of the recombinant protein generally is very expensive rather than the preparation of a small molecular substance. Moreover, there are many restrictions as a medical drug in terms of physical properties or administration methods due to its proteinic characteristics. Considering these points, a low molecular organic compound having the equal activity to that of said BMP protein, if any, will become a highly promising medical drug.
For such an exploring method, an example was so far reported using a murine BMP-2 promoter (W097/15308). The region of the murine BMP-2 promoter has been already cloned (Biochem. Biophys. Acta, Vol. 1218, p. 221-224, 1994), but no report has been published for the region of human BMP-2 promoter and an exploring method employing the human BMP-2 promoter. A comparison of the DNA sequence of 741 bp of the 5xe2x80x2 upstream exon region of said murine BMP-2 promoter with the corresponding human BMP-2 promoter sequence (the base sequence No. from 4709 to 5483 shown in SEQ ID NO. 1 of the Sequence Listing) of the present invention shows 71.6% homology between these two sequences. Because all the materials used for establishing the exploring method presented by the present invention are of human origin, the substance discovered from exploration can be quite effective in clinical application.
The present invention provides a 5xe2x80x2 upstream region DNA containing a promoter of human BMP-2. By using 5xe2x80x2 upstream region gene containing the human BMP-2 promoter and an animal cell introduced with the recombinant expression vector which has been connected to an appropriate reporter gene, the low molecular weight compounds which regulate positively or negatively the expression of human BMP-2 can be explored with reference to a reporter activity. The low molecular weight compounds and their derivatives have morphogenetic activity and inhibiting activity for bone and cartilage through the expression of human BMP-2 and are useful as preventive or therapeutic agents for bone and cartilage diseases, remedies for osteometastasis, or therapeutic and preventive agents for osteohyperplasia.